To be able to study the structure of biological specimens such as tissues or cells with an electron microscope, ultra-thin sections only a few nm in size are prepared and placed on object support screens made of metal, preferably of nickel (in the following referred to as small metal screens). For electron-microscopic studies, the sections are rendered contrasty or individual constituents of the specimen are marked by use of special or cytochemical methods. These cytochemical methods are often based on the principle of ligand pair formation, the first ligand possibly being present in the biological specimen and the second ligand, upon coming in contact with this specimen, binding as binding partner to the first ligand. Examples of biologically based ligand pairs are antigen/antibody binding pairs, enzyme/substrate binding pairs, lecithin/sugar, hormone/receptor systems, DNA/DNA pairs and DNA/RNA pairs.
The prior art discloses numerous methods involving the antigen/antibody binding pair. These methods are collectively referred to as immunohistochemistry and immunocytochemistry (in the following referred to as marking techniques). For example, U.S. Pat. No. 5,143,714 discloses a method whereby an antigen is adsorbed from a liquid specimen into a pelletizable gel substance. The gel pellet is surrounded by a diffusion barrier, integrated as a block into a punched-out gel matrix and subsequently, as a tissues specimen, subjected to immunological marking techniques. DE 38 78 167 T2 describes the use of colloidal gold particles for the marking of ligands by the immunogold staining technique. A greatly improved method permitting qualitative and quantitative evaluation of an antigen in a specimen is disclosed in U.S. Pat. No. 5,079,172 as a sandwich assay whereby the antigen-binding first antibody is marked with a gold-labeled second antibody that binds to the first antibody. By the electron-microscopic evaluation method, the antigen in the specimen can be determined qualitatively and quantitatively from the amount of gold particles.
Many procedures of immunohistochemistry and immunocytochemistry for immunological marking of thin-sectioned tissues have in common the feature that in most cases they consist of 10 to 20 individual processing steps. In a large number of cases, the processing steps consist of washing the specimen to be studied with buffering or marking solutions.
Such washing is currently carried out manually by an expensive procedure whereby individual drops of the aqueous buffer solution or marking solution are placed onto a hydrophobic substrate (for example PARAFILM®, PARLODION®, Colloidion or FORMFAN®). The small metal screens with the thin-sectioned tissue are placed individually onto the drops to enable the tissue to react with the treatment liquid. Because of the light weight of the small metal net and the surface tension of the liquid drop, the small metal net floats on the surface of the drop. After a certain contact time for this step (often 5–10 min), the small metal net is moved on to the next drop by use of tweezers. This is continued all the way to the last position of the standard procedure tying down an operator for up to several hours per immunological marking reaction.
It can readily be seen that this manual process requires the continuous attention of the working personnel and that the labor costs are high because of the large amount of time involved. The number of specimens that can be worked on at the same time is very limited, and errors by the working personnel during the exact pipetting and positioning of liquid drops of very small volume cannot be excluded. The manual method cannot exclude mixing up the specimens after the long treatment period during the immunological marking, but this could be prevented by use of a specimen carrier with identification in the form of a chip or bar code, as described in Utility Model DE 299 06 382 U1.
Furthermore, the evaporation of the liquid drops during prolonged standard procedures presents a major problem.
Although Utility Model DE 298 17 912 U1 discloses a device for washing preparations to be subjected to microscopy on carriers following immunochemical treatment, this device is a washing box in which a major quantity of washing solution flows over the preparation and the carrier at a certain flow rate. Such a device is not suited for immunological marking techniques, because the antibody-containing marking solutions used are very expensive, and hence, are used in only very small volumes. To date, no device or method is known that allows an automated execution of immunological marking techniques for thin-sectioned tissues.